The objective of this research project include: (a) The isolation and characterization of ts mutants of MSV. Candidate ts mutants will be further analyzed in terms of adequate thermosensitivity and reversion rate in order to judge their experimental usefulness. Mutagenized MSV stocks will continue to be screened for presumptive ts mutants. (b) An analysis of polypeptides of cells transformed by wild-type MSV and a cold-sensitive mutant of MSV. Proteins solubilized from uninfected NRK cells, NRK cells transformed by wild-type MSV or a cs mutant of MSV, and revertants of MSV transformed cells will be analyzed by two-dimensional gel electrophoresis. The extent and specificity of polypeptide changes as determined by Coomassie Blue staining or radio-labeling will be correlated with expression of the transformed phenotype. As part of an ongoing collaboration with Drs. T. Wood and R. B. Arlinghaus, at the Biology Department, M. D. Anderson Hospital and Tumor Institute, a 63,000 dalton polypeptide (Pr63), precipitated by anti-p30 serum but not by anti-gp69/71 serum, was identified in extracts of NRK cells infected with the cold-sensitive mutant of MSV. The Pr63 band will be characterized as to its relationship to the various viral gene products by both immunological techniques using monospecific antisera to each of the viral proteins and tryptic peptide analysis. (c) An analysis of genomic RNA of defective and "nondefective" strains of MSV. Analysis of the size of genomic RNA extracted from MLV and MSV-MLV will be continued and expanded to include replication-defective MSV and MSV produced by the morphological revertant of MSV-transformed NRK cells. The size of the viral RNA subunits will be analyzed by agarose-urea gel electrophoresis. Based on this type of analysis, it should be possible to correlate functional viral defects with viral RNA deletions.